High-performance liquid chromatography has been applied to determination of modifier activity in (\alpha)-lactalbumin ( (\alpha)-LA). An amino-bonded column separates uridine diphosphate (UDP) (product), UDPgalactose (substrate), and uridine monophosphate (UMP). From an aliquot of the same sample, a column for carbohydrate analysis separates lactose (the other product) and galactose-1,2-cyclic phosphate (Gal-c-P). Nucleotide peaks are detected by measurement of (\boldsymbol{A}_{262}) and those of carbohydrate by ({ }^{3} \mathrm{H}) counting, the isotope orig. inating from UDP-galactose- ({ }^{3} \mathrm{H}). A pH of 6.3 was taken as optimal for production of UDP since, at this level, the unwanted side reaction is minimized, by which UMP and Gal-c-P are formed. Thus, the conservation of substrate so effected may have contributed to an enhanced production of UDP. The reaction by which UDP and lactose are produced was linear for (120 \mathrm{~min}), as followed by UDP formation, but it continued to at least (300 \mathrm{~min}). Production of lactose was equivalent to that of UDP, when (\alpha)-LA was the modifying protein. From a survey of seven other proteins, only lysozyme and ovalbumin showed ability to produce UDP. However, failure of the last two proteins to produce lactose indicates absence of modifier activity and demonstrates the need for monitoring both products. ") 1995 Academic Press, Inc.